Biotransformation of soybean phytosterols into 9α-hydroxy-4-androstene-3,17-dione(9-OHAD)by mycobacteria is the core step in the synthesis of adrenocortical hormone.However,the low permeability of the dense cell enve...Biotransformation of soybean phytosterols into 9α-hydroxy-4-androstene-3,17-dione(9-OHAD)by mycobacteria is the core step in the synthesis of adrenocortical hormone.However,the low permeability of the dense cell envelope largely inhibits the overall conversion efficiency of phytosterols.The antigen 85(Ag85)complex encoded by fbpA,fbpB,and fbpC was proposed as the key factor in the combined catalysis of mycoloyl for producing mycolyl-arabinogalactan(m-AG)and trehalose dimycolate(TDM)in mycobacterial cell envelope.Herein,we confirmed that fbpC3 was essential for the biotransformation of trehalose monomycolate(TMM)to TDM in Mycolicibacterium neoaurum.The deficiency of this gene raised the cell permeability,thereby enhancing the steroid uptake and utilization.The 9-OHAD yield in the fbpC3-deficient 9-OHAD-producing strain was increased by 21.3%.Moreover,the combined deletion of fbpC3 and embC further increased the 9-OHAD yield compared to the single deletion of fbpC3.Finally,after 96 h of bioconversion in industrial resting cells,the 9-OHAD yield of 11.2 g/L was achieved from 20 g/L phytosterols and the productivity reached 0.116 g/L/h.In summary,this study suggested the critical role of the fbpC3 gene in the synthesis of TDM in M.neoaurum and verified the feasibility of improving the bioconversion efficiency of phytosterols through the cell envelope en-gineering strategy.展开更多
Treatment of Mycobacterium abscessus(Mab)infections is very challenging due to its intrinsic resistance to most available drugs.Therefore,it is crucial to discover novel anti-Mab drugs.In this study,we explored an int...Treatment of Mycobacterium abscessus(Mab)infections is very challenging due to its intrinsic resistance to most available drugs.Therefore,it is crucial to discover novel anti-Mab drugs.In this study,we explored an intrinsic resistance mechanism through which Mab resists echinomycin(ECH).ECH showed activity against Mab at a minimum inhibitory concentration(MIC)of 2μg/ml.A embC strain in which the embC gene was knocked out showed hypersensitivity to ECH(MIC:0.0078-0.0156μg/ml).The MICs of ECH-resistant strains screened with reference to AembC ranged from 0.25 to 1μg/ml.Mutations in EmbB,including D306A,D306N,R350G,V555l,and G581S,increased the Mab's resistance to ECH when overexpressed in AembC individually(MIC:0.25-0.5μg/ml).These EmbB mutants,edited using the CRISPR/Cpf1 system,showed heightened resistance to ECH(MIC:0.25-0.5μg/ml).The permeability of these Mab strains with edited genes and overexpression was reduced,as evidenced by an ethidium bromide accumulation assay,but it remained significantly higher than that of the parent Mab.In summary,our study demonstrates that ECH exerts potent anti-Mab activity and confirms that EmbB and EmbC are implicated in Mab's sensitivity to ECH.Mutation in EmbB may partially compensate foralossof EmbCfunction.展开更多
基金the National Natural Science Foundation of China(Nos.21776075 and 32100067)the Natural Science Founda-tion of Shanghai(No.20ZR1415100)+2 种基金the National Key Research and Development Program of China(No.SQ2020YFC210061)the China Postdoctoral Science Foundation(No.2020M671028),the Shanghai Municipal Health Commission(No.20204Y0380)the Teacher’s Professional Development Project of Shanghai Municipal Education Commission,and the Scientific Research Foundation of SUMHS.
文摘Biotransformation of soybean phytosterols into 9α-hydroxy-4-androstene-3,17-dione(9-OHAD)by mycobacteria is the core step in the synthesis of adrenocortical hormone.However,the low permeability of the dense cell envelope largely inhibits the overall conversion efficiency of phytosterols.The antigen 85(Ag85)complex encoded by fbpA,fbpB,and fbpC was proposed as the key factor in the combined catalysis of mycoloyl for producing mycolyl-arabinogalactan(m-AG)and trehalose dimycolate(TDM)in mycobacterial cell envelope.Herein,we confirmed that fbpC3 was essential for the biotransformation of trehalose monomycolate(TMM)to TDM in Mycolicibacterium neoaurum.The deficiency of this gene raised the cell permeability,thereby enhancing the steroid uptake and utilization.The 9-OHAD yield in the fbpC3-deficient 9-OHAD-producing strain was increased by 21.3%.Moreover,the combined deletion of fbpC3 and embC further increased the 9-OHAD yield compared to the single deletion of fbpC3.Finally,after 96 h of bioconversion in industrial resting cells,the 9-OHAD yield of 11.2 g/L was achieved from 20 g/L phytosterols and the productivity reached 0.116 g/L/h.In summary,this study suggested the critical role of the fbpC3 gene in the synthesis of TDM in M.neoaurum and verified the feasibility of improving the bioconversion efficiency of phytosterols through the cell envelope en-gineering strategy.
基金This work was supported by the National Key R&D Program of China(2021YFA1300900)the National Natural Science Foundation of China(21920102003,82022067,and 22037006)+5 种基金the Chinese Academy of Sciences Grants(154144KYSB20190005 and YJKYYQ20210026)the Key R&D Program of Sichuan Provenience(2023YFSY0047)the State Key Laboratory of Respiratory Disease,Guangzhou Institute of Respiratory Diseases,First Affiliated Hospital of Guangzhou Medical University(SKLRD-Z-202414,SKLRD-OP-202324,SKLRD-Z-202301,SKLRD-OP-202113,and SKLRD-Z-202412)Guangzhou Scienceaand Technology Plan-Youth Doctoral"Sail"Project(2024A04J4273)President's International Fellowship Initiative-CAS(2023VBC0015)the National Foreign Young Talent Program(QN2022031002L).
文摘Treatment of Mycobacterium abscessus(Mab)infections is very challenging due to its intrinsic resistance to most available drugs.Therefore,it is crucial to discover novel anti-Mab drugs.In this study,we explored an intrinsic resistance mechanism through which Mab resists echinomycin(ECH).ECH showed activity against Mab at a minimum inhibitory concentration(MIC)of 2μg/ml.A embC strain in which the embC gene was knocked out showed hypersensitivity to ECH(MIC:0.0078-0.0156μg/ml).The MICs of ECH-resistant strains screened with reference to AembC ranged from 0.25 to 1μg/ml.Mutations in EmbB,including D306A,D306N,R350G,V555l,and G581S,increased the Mab's resistance to ECH when overexpressed in AembC individually(MIC:0.25-0.5μg/ml).These EmbB mutants,edited using the CRISPR/Cpf1 system,showed heightened resistance to ECH(MIC:0.25-0.5μg/ml).The permeability of these Mab strains with edited genes and overexpression was reduced,as evidenced by an ethidium bromide accumulation assay,but it remained significantly higher than that of the parent Mab.In summary,our study demonstrates that ECH exerts potent anti-Mab activity and confirms that EmbB and EmbC are implicated in Mab's sensitivity to ECH.Mutation in EmbB may partially compensate foralossof EmbCfunction.
